The Role Of STAT3-regulated Autophagy In Angiotensin?-induced Senescence Of Renal Tubular Epithelial Cells And The Role Of Losartan On It

Objective:The aging is the current global demographic trend,which brings many challenges to the social medical care,pension,labor force,and economic development in the world.Therefore,the study of aging-related diseases is of great significance.The kidney has the important functions of excretion of metabolic wastes and maintaining the balance of water and electrolytes.The effect of aging on the body cannot be ignored.Kidney aging includes physiological aging caused by age and stress-induced premature aging due to some diseases,such as hypertension and diabetes.It can increase the incidence of chronic kidney disease and bring heavy medical burden to society.In the tissue level,aging is manifested as the aggregation of biological macromolecules such as DNA,protein and lipids,organelle damage,and toxic modification;autophagy is a cellular pathway involving the degradation of proteins and organelles,which can maintain cell homeostasis and participate many pathophysiological processes such as aging,inflammation,tumors,and immunity.STAT3 as an important member of JAK/STATs pathway,is involved in the occurrence of various kidney diseases,autophagy,aging and other processes.The renin-angiotensin-aldosterone system is the main system that maintains the normal physiological functions of the kidney,and its abnormality can also lead to the occurrence of a variety of kidney diseases.Angiotensin II(Ang II)is the main effector molecule.Mechanistically,the role of STAT3-regulated autophagy in Ang II-induced senescence of renal tubular epithelial cells has not been elucidated.Therefore,the aim of this study was to investigate the role of STAT3-regulated autophagy in Ang II-induced senescence of renal tubular epithelial cells and the role of losartan on it,with a view to providing strategies for delaying kidney aging and preventing and treating chronic kidney disease.Methods:1.Establish Ang II-induced senescence model of human renal tubular epithelial cells and observe autophagy in the senescence process.10^-6mol/L Ang II treatment HK-2 cells for 0-72h,and detect cell cycle by flow cytometry,Western blot detect the expression level of senescence-related protein P21,?-galactosidase staining detect the positive rate of senescent cells.Then,Ang II and 3-MA were applied to HK-2 cells,and western blot detect the expression levels of autophagy-related proteins LC3II and P62,aging-related protein P21,electronmicroscopy detect the number of autophagosomes,LC3-GFP-m RFP autophagy double-labeled adenovirus transfection observe the number of autophagy spots,?-galactosidase staining to detect the positive rate of senescent cells,to observe the changes and effects of Ang II-induced autophagy in the process of senescence in renal tubular epithelial cells.2.The role of STAT3 in the regulation of autophagy on Ang II-induced senescence of renal tubular epithelial cells and the role of Losartan on it.In the proess of Ang II-induced senescence of renal tubular epithelial cells,western blot was used to detect the changes of STAT3/PI3KC3 pathway.Then,S3I-201 and si RNA-STAT3 gene blocking STAT3 were used to observe p STAT3 protein,senescence-related protein P21,autophagy-related protein LC3II and P62 expression levels by western blot,electronmicroscopy to detect the number of autophagosomes,LC3-GFP-m RFP autophagy double-labeled adenovirus transfection to observe the number of autophagy spots,?-galactosidase staining to detect the positive rate of senescent cells.Losartan was used to treat HK-2 cells during induced by Ang II,and western blot was used to observe the expression levels of p STAT3,P21,LC3II and P62proteins,electronmicroscopy to detect the number of autophagosomes,LC3-GFP-m RFP autophagy double-labeled adenovirus transfection to observe the number of autophagy spots,?-galactosidase staining to detect the positive rate of senescent cells.3.In vivo verification of the changes of STAT3 pathway,autophagy and senescence of renal tubular epithelial cells in mice kidney induced by Ang II and the role of losartan on it.After 18 male C57BL/6J mice were adaptively reared for 1 week,the mice were randomly divided into 3 groups,the young control group,the Ang II group(infusion of Ang II,1000 ng/kg/min for 4 weeks),Ang II+Losartan group(infusion of Ang II+intragastric losartan at 30 mg/kg/d by intragastric gavage for 2 weeks).Each group was 6mice.The blood pressure of the mice were monitored weekly.After the end of the 4th week,blood and urine of mice were collected and blthe changes of ood urea nitrogen,blood creatinine,24h urine protein,and urine NAG were detected.Kidney tissue were collected and used Masson staining,?-galactosidase staining,and immunohistochemistry.Results:1.Ang II induces the enhancement of autophagy activity and mediates the senescence of human renal tubular epithelial cells.Treatment of human HK-2 cells with Ang II at the concentration of 10^-6mol/L,cell cycle testing reveals that Ang II acts on HK-2 cells After 72h,more than 80%of the cells entered the G₀-G₁phase,which was significantly higher than that of the normal control group;western blot results showed that compared with the normal control group,the expression of HK-2 cell P21 protein began to increase significantly at 48h and reached a peak at 72h;?-galactosidase staining found that,the positive rate of?-galactosidase staining cells in HK-2 cells increased significantly at 48h,and reached a peak at 72h.During the senescence of HK-2 cells by Ang II-induced,western blot showed that the expression of autophagy-related protein LC3II increased significantly at 48h and reached a peak at 72h,and the expression of P62 protein began to decrease significantly at 24h and reached the lowest at 72h.Electronmicroscopy showed that the number of autophagosomes in HK-2 cells increased significantly at 72h;LC3-GFP-m RFP autophagy double-labeled adenovirus transfection showed the number of LC3fluorescence spots in HK-2 cells significant increase at 72h.After adding 3-MA to inhibit autophagy of human HK-2 cells,compared with the Ang II group(72h),the Western blot results of the Ang II+3-MA group showed that the expression of LC3II protein was significantly decreased,the expression of P62 protein was significantly increased,and the expression of P21 protein was significantly decreased.The number of autophagosomes detected by electronmicroscopy was significantly reduced,the number of LC3fluorescent spots transfected with LC3-GFP-m RFP autophagy double-labeled adenovirus was significantly reduced,and the positive rate of?-galactosidase stained cells was significantly reduced.2.Ang II induces activation of the STAT3/PI3KC3 pathway during the senescence of human renal tubular epithelial cells.Drug inhibition and gene silencing STAT3 expression can inhibit the autophagy activation of Ang II-induced human renal tubular epithelial cells,and reduce cellular senescence.10^-6mol/L Ang II treat human HK-2 cells.Western blot showed that the expression of p STAT3 protein increased significantly at 24h,with the highest expression at 72h;the expression of PI3KC3 protein increased significantly at 48h and the highest expression at 72h.After adding S3I-201,si RNA-STAT3 and losartan to human HK-2cells with 10^-6mol/L Ang II,compared with Ang II group(72h),Ang II+S3I-201 group,Ang II+si RNA-STAT3 group and Ang II+Losartan group western blot showed the expression of p STAT3 protein was significantly inhibited,the expression of LC3II protein was significantly reduced,the expression of P62 protein was significantly increased,and the expression of P21 protein was significantly decreased.Electronmicroscopy showed that the number of autophagosomes was significantly reduced.LC3-GFP-m RFP autophagy double-labeled adenovirus transfected LC3fluorescent spots were significantly reduced.The positive rate of?-galactosidase staining was significantly reduced.3.Losartan can inhibit the activation of STAT3 and autophagy during kidney aging induced by Ang II in mice,and delay the senescence of renal tubular epithelial cells in mice.With the increase of age,compared with the young control group,the systolic and diastolic blood pressures of the Ang II group mice were gradually increased.At the 4th week of age,the systolic and diastolic blood pressures of the Ang II group mice were significantly higher than those of the young control group.Blood urea nitrogen,serum creatinine,24h urine protein,and urine NAG were significantly higher than the young control group.Masson staining showed that the degree of tubular interstitial fibrosis was significantly increased,and the positive rate of?-galactosidase staining was significantly increased.Immunohistochemistry showed the expression level of P21 increased significantly,and the expression levels of p STAT3 and LC3 were also significantly higher than the young control group in renal tubular epithelial cells.After the application of losartan,at the 4th week of age,compared with the Ang II group,the systolic blood pressure,blood urea nitrogen,24-hour urine protein and urine NAG of the Ang II+Losartan group decreased significantly,but the decrease of diastolic blood pressure and serum creatinine were no significant difference;Masson staining showed that the degree of tubular interstitial fibrosis was significantly reduced;the positive rate of?-galactosidase staining was significantly reduced;immunohistochemistry showed the expression of P21 in renal tubular epithelial cells was significantly reduced,and the expression of p STAT3 and LC3 was also significantly reduced.Conclusion:1.Ang II can induce the senescence of human renal tubular epithelial cells by promoting autophagy;Using 3-MA to inhibit autophagy can reduce the senescence of human renal tubular epithelial cells induced by Ang II.2.Ang II induces activation of the STAT3/PI3KC3 pathway during the senescence of human renal tubular epithelial cells;S3I-201,si RNA-STAT3 and losartan can inhibit the activation of STAT3,thereby inhibiting the autophagy of human renal tubular epithelial cells induced by Ang II and reducing cell senescence.3.STAT3 pathway and autophagy activation are involved in the senescence of mouse renal tubular epithelial cells induced by Ang II;Losartan can inhibit the activation of STAT3 and autophagy,delaying the mouse renal tubular epithelial cells senescence.This study provides in vivo verification support for the first and second parts of the paper.